FLUDARABINE TRIPHOSPHATE INHIBITS NUCLEOTIDE EXCISION-REPAIR OF CISPLATIN-INDUCED DNA-ADDUCTS IN-VITRO

Fludarabine arabinofuranosyl-2-fluoroadenine-5'-monophosphate) is clin ically active against chronic lymphocytic leukemia and low-grade lymph omas, We reported previously that fludarabine nucleoside synergistical ly enhanced cisplatin (CDDP)-induced cytotoxicity in vitro, and that t he synergism was concomitant with inhibition of removal of cellular CD DP-induced DNA interstrand cross-links, which are presumably repaired by homologous recombinational repair, To extend our work, we investiga ted whether fludarabine inhibits nucleotide excision repair (NER) of C DDP-induced DNA intrastrand adducts, The effect of fludarabine on NER was determined using a cell-free system in which a plasmid containing the DNA adducts served as the substrate for repair enzymes in whole-ce ll extracts from repair-competent cells, To prevent the cell-bound hig h mobility group box-containing proteins from interfering with repair, cell extracts were depleted with high mobility group hox proteins by immunoprecipitation prior to the assay, Repair synthesis, measured by the incorporation of [P-32]dATP or [P-32]dCTP, was inhibited by 50% at 26 or 43 mu M fludarabine triphosphate, respectively; the effect was dose dependent and may have resulted from the termination of repair-pa tch elongation, These results were consistent with those from pulse-ch ase experiments demonstrating the conversion of nicked circular plasmi d to the closed circular form by cell extracts filling the repair gaps , When proliferating cell nuclear antigen-depleted cell extracts were used and aphidicolin was added in the repair assay to arrest NER at th e incision/excision stage, 100 mu M fludarabine triphosphate inhibited about 55% of the conversion of nicked plasmids from the closed circul ar damaged plasmid substrate; the inhibition was dose dependent, We co nclude that fludarabine triphosphate inhibited NER at the steps of inc ision and repair synthesis, These results suggest that fludarabine may serve as a potential repair modulator to improve the antitumor effica cies of combination regimens containing agents that induce NER.